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Graphical Abstract
Investigation of pharmacokinetic properties of CK2 Inhibitors with an Indeno[1,2-b]indole scaffold
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t [h] confluence [%]
Investigation of pharmacokinetic properties of CK2 Inhibitors with an - - PowerPoint PPT Presentation
Investigation of pharmacokinetic properties of CK2 Inhibitors with an - - PowerPoint PPT Presentation
Investigation of pharmacokinetic properties of CK2 Inhibitors with an Indeno[1,2- b ]indole scaffold Robin Birus 1, *, Marc Le Borgne 2 , and Joachim Jose 1 1 Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Corrensstrasse 48,
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Abstract:
The highly pleiotropic and constitutively active protein kinase CK2 plays an important role in several cellular
- mechanisms. Due to its overexpression and elevated activity in tumor cells, CK2 became an important target in
tumor therapy nowadays. It was shown, that the kinase causes antiapoptotic and proliferation enhancing effects in neoplastic tissues [1,2]. Moreover, the reduction of CK2 activity in tumor cells leads to apoptosis while normal cells stay unaffected [3]. Indeno[1,2-b]indoles are ATP competitive CK2 inhibitors with IC50 values in the nanomolar range of concentration. NA16 was described as one of the most potent indeno[1,2-b]indoles with an IC50 value of 25 nM [4]. Therefore, the pharmacokinetic properties of NA16 were further analyzed during this study. It could be shown that NA16 reduces the growth of different tumor cell lines and induces cancer cell apoptosis. Furthermore, its effect on HUVEC cell growth was comparable with the effect of CX-4945, a CK2 inhibitor in clinical trials [5], which was used as control. Intracellular concentrations of NA16 were higher than concentrations of CX-4945 at different time points. Metabolism studies showed that NA16 is moderate metabolic stable and is not glucuronidated. These results underline the potential of NA16 as an antitumor drug. References: [1] Ahmed, K et al.: Trends Cell Biol 2002, 12, 226-230. [2] Meggio, F. and Pinna, L.A.: FASEB J. 2003, 17, 349-368. [3] Slaton, J. W. et al.: Mol Cancer Res 2004, 2, 172. [4] Gozzi, G. J. et al.: J Med Chem 2015, 58, 265-277. [5] Siddiqui-Jain, A. et al.: Cancer Res 2010, 70, 24.
Keywords: Protein kinase CK2; ATP-competitive inhibitor; cell culture; pharmacokinetic
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Introduction
- CK2:
- Heterotetrameric holoenzyme
- Ubiquitous Ser/Thr kinase
- Constitutively active; highly pleiotropic
- Overexpression and higher activity in tumor cells
- Important target in tumor therapy
- Indeno[1,2-b]indoles:
- Potent, ATP competitive CK2 inhibitors
- Low IC50 values: <1 µM
- NA16: One of the most potent Indeno[1,2-b]indole
derivatives ➢ IC50 = 25 nM
NA16
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Results and discussion
- 1. Influence of NA16 on tumor cell growth
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A549
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40 µM 35 µM 30 µM 25 µM 20 µM 15 µM 10 µM 5 µM 1 µM DMSO 1% t [h] confluence [%]
LNCaP
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30 µM 15 µM 12.5 µM 10 µM 7.5 µM 3.75 µM 1.88 µM 0.94 µM 0.47 µM DMSO 1% t [h] confluence [%]
A431
12 24 36 48 20 40 60 80 100
30 µM 15 µM 12.5 µM 10 µM 7.5 µM 3.75 µM 1.88 µM 0.94 µM 0.47 µM DMSO 1% t [h] confluence [%]
- Investigation of tumor cell growth via Live Cell Imaging
- Analyzing the effect of NA16 on growth of different cancer cell lines: A431 (epidermal),
A549 (lung) and LNCaP (prostate)
- NA16 reduces growth of all tested tumor cell line dose-dependently
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Results and discussion
- 1. Influence of NA16 on tumor and normal cell growth
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CX-4945 NA16 10 20 30
A431 A549 LNCaP HUVEC EC50 [µM]
- EC50: Inhibitor concentration
which causes a reduction of cell growth by 50% compared to cells treated with 1% DMSO
- HUVEC: Human umbilical vein
endothelial cell ➢ Non-cancer cell line
- NA16 reduces tumor cell
growth in the same dimension as CX-4945
- EC50 of NA16 on HUVEC cells
comparable to EC50 of CX-4945
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Results and discussion
- 2. Induction of apoptosis by NA16
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DMSO 1% NA16 20 µM 0 h 48 h 24 h
12 24 36 48 5000 10000 15000 20000 25000
CX-4945 1 µM CX-4945 10 µM CX-4945 20 µM NA16 1 µM NA16 10 µM NA16 20 µM DMSO 1% t [h] Green Object Count (1/Well)
- Apoptosis analysis of A431 cells via Live Cell
Imaging
- Fluorescence labeling of apoptotic cells after
treatment with CK2 inhibitors ➢ Induction of apoptosis detectable as well after treatment with CX-4945 as with NA16
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Results and discussion
- 3. Cellular uptake
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Intracellular concentrations
CX-4945 NA16 100 200 2000 4000 6000
1 h 5 h 12 h c [nM]
- Uptake analysis of CK2 inhibitors into
A431 cells
- Extracellular concentration: 1 µM
- Different incubation times: 1 h, 5 h, 12 h
- Cell lysis and sample purification
- Quantification of intracellular inhibitor
concentration via HPLC-MS/MS
- c (NA16) > c (CX-4945)
- NA16: Decrease of intracellular
concentration
- CX-4945: Increase of intracellular
concentration
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Results and discussion
- 4. Metabolism studies
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- Investigation of metabolic stability of NA16:
- Quantification of intact NA16 after
incubation with phase I metabolism enzymes via HPLC-MS (Börgel et al. 2019)
- Comparison with imipramine (control
for metabolic labile substance)
- Amount of intact NA16 higher than amount
- f imipramine
➢ NA16 has a moderate metabolic stability
Imipramine NA16 20 40 60 80
Amount of intact parent [%]
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Results and discussion
- 4. Metabolism studies
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- Metabolite identification:
- NA16 + phase I metabolism enzymes vs. NA16 + phase I & II metabolism enzymes
(glucuronidation was investigated representative for phase II metabolism)
- Analysis of metabolites via HPLC-MS (Börgel et al. 2019)
- Metabolites in both samples were equivalent
- No glucuronidation products of NA16 detectable
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Conclusions
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- Effects of NA16:
- Reduction of growth of A431, A549 and LNCaP
cells
- Influence on HUVEC growth comparable with
influence of CX-4945
- Induction of apoptosis weaker compared to
treatment with CX-4945
- Intracellular concentration higher than
concentration of CX-4945; time-dependent decrease
- NA16 is moderate metabolic stable; no
glucuronidation products detectable ➢ NA16 is worth analyzing further effects concerning its anti-tumor activity
I m i p r a m i n e N A 1 6 20 40 60 80 Amount of intact parent [%]
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